Project Summary/Abstract Research towards a cure for human immunodeficiency virus (HIV) and mitigation of the impact of HIV/ tuberculosis co-infections are NIH priorities. Both research areas rely on studies of macaque monkeys experimentally infected with simian immunodeficiency virus (SIV). Interventions to elicit effective CD8+ T cell responses to SIV and Mycobacterium tuberculosis (MTb) in macaques are under active develop- ment. These studies are limited by the dearth of SIV- and MTb-specific CD8+ T cell responses restricted by common macaque major histocompatibility complex (MHC) class I alleles. We began addressing this problem in the previous grant period and identified dozens of novel CD8+ T cell responses in SIV and MTb, as well as in Zika virus, ebolavirus, and influenza. We also realized that conventional T cell epitope discovery and characterization is unwieldy, slow, and cumbersome. Consequently, we demonstrated that MHC class I binding to millions of peptides can be measured si- multaneously, providing a transformative and extremely rapid way to define CD8+ T cell epitopes. The purpose of this competitive revision is to use this new technology to define CD8+ T cell epitopes in SIV and MTb restricted by 16 common macaque MHC class I alleles. Specifically, we will: Aim 1: Identify SIV and MTb CD8+ T cell responses restricted by 16 common macaque MHC class I molecules. We will assess peptide binding of each 8-, 9-, 10, and 11-mer peptide in the pro- teomes of every SIV and SHIV genome currently in Genbank and the MTb Erdman strain using an ul- tradense peptide array. MHC:peptide tetramers will be produced for responses that are experimentally validated. Aim 2: Define peptide binding motifs for the 16 common MHC class I molecules by determining the impact of every amino acid substitution at each residue in CD8+ T cell epitopes on peptide binding. The peptide binding motifs can be used to improve algorithms for in silico prediction of MHC:peptide binding. Research resources from this study will be made available to the research community through real-time sharing of peptide array data, deposition of experimentally validated CD8+ T cell epitopes in the Immune Epitope Database, and distribution of MHC:peptide tetramers through the NIH Tetramer Core Facility. Furthermore, the definition of CD8+ T cell epitopes using ultradense peptide arrays is generalizable and could revolutionize the identification of pathogen-specific epitopes in all species, including humans.